Anti hk2 22029 1 ap

Anti-mouse CD3e (100340), anti-mouse CD28 (102116), recombinant mouse IL-2 (575406), recombinant mouse IL-6 (575702), recombinant mouse TGF-β (763102), recombinant mouse IL-12 (577004), recombinant mouse IL-23 (589002), Brilliant Violet 421-conjugated anti-mouse CD4 (100443), PerCP anti-mouse CD8a (100731), PE-conjugated anti-mouse/human CD44 (103008), APC-conjugated anti-mouse CD62L (104412), PE/Cyanine7-conjugated anti-mouse IFN-γ (505826), Brilliant Violet 421-conjugated anti-mouse IL-17A (512321), PE-conjugated anti-mouse IL-17A (506904), Alexa Fluor^® 647 anti-mouse/rat/human FOXP3 (320014), FITC anti-mouse CD11c (117306), Brilliant Violet 421™ anti-mouse I-A/I-E (107620), PE/Cy7 anti-mouse CD86 (105014), and PE anti-mouse F4/80 (123110) were purchased from Biolegend (San Diego, CA, USA). Anti-phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) (4228S), anti-PI3 Kinase p85 (4292S), anti-phospho-AKT (Ser473) (D9E) XP^® Rabbit mAb (4060S), anti-AKT (9272S), anti-PKM2 (4053S) and anti-β-Actin (3700S) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA); anti-PGK1 (A12686), anti-ENO1 (A11448), and anti-LDHA (A1146) antibodies were got from Abclonal (Wuhan, China); anti-HK2 (22029-1-AP) and anti-Glut1 (21829-1-AP) antibodies were obtained from Proteintech (Wuhan, China); and 740 Y-P (HY-P0175) was ordered from Medchem Express (Shanghai, China).

Cells were lysed in RIPA lysis buffer (Beyotime, China) for 30 min on ice, followed by centrifugation at 12,000 g at 4°C for 10 min. The supernatant was harvested and protein concentration was measured with BCA Assay Kit (Beyotime, China). Proteins were separated on 10% SDS-PAGE gels and then transferred to PVDF membranes. After blocking in 5% skimmed milk for about 1 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The membranes were visualized in the Bio-Rad gel image analysis system after incubation with corresponding second antibodies on the next day. The following primary antibodies were used in our study: anti-β-actin (A1978, Sigma-Aldrich), anti-HK2 (22029-1-AP, Proteintech), anti-PKM2 (15822-1-AP, Proteintech).

Cells were lysed on ice for 30 min in RIPA lysis buffer supplemented with protease inhibitors and a phosphatase inhibitor cocktail (Bimake), followed by centrifugation at 12,000 g for 10 min. The protein concentration was measured using a BCA Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking, membranes were incubated with primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. The immunoblots were visualized by ECL chemiluminescence (GE healthcare, Buckinghamshire, UK) using a Bio-Rad gel image analysis system. The following primary antibodies were used in this study: anti-ETV4 (sc-113, Santa Cruz), anti-HK2 (22029-1-AP, Proteintech), anti-LDHA (19987-1-AP, Proteintech), anti-PDK1 (3062 T, Cell Signaling Technology), anti-c-MYC (10828-1-AP, Proteintech), anti-OCT4 (11263-1-AP, Proteintech), anti-NANOG (14295-1-AP, Proteintech), anti-LIN28 (11724-1-AP, Ptoteintech), anti-SHH (ab53281, Abcam), anti-GLI1 (66905-1-Ig, Proteintech), anti-CXCR4 (60042-1-Ig, Proteintech), anti-β-actin (A1978, Sigma-Aldrich).